oxBS-seq publications

Publications utilising CEGX technology

The following is a list of high impact publications utilising oxidative bisulfite to study DNA (hydroxy)methylation in various fields including cancer biology, development, stem cell research, and neuroscience.

2018 oxBS papers:

Kernaleguen et al. Whole-genome bisulfite sequencing for the analysis of genome-wide DNA methylation and hydroxymethylation patterns at single-nucleotide resolution. Methods Mol Biol. 2018;1767:311-349 doi: 10.1007/978-1-4939-7774-1_18.
The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and disease-associated investigations. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered as the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here we provide two detailed protocols based on commercial kits for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. If only DNA methylation is of interest, sequencing libraries can be constructed from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For samples with significant levels of hydroxymethylation such as stem cells or brain tissue, we describe the protocol of oxidative bisulfite sequencing (OxBs-seq), which in its current version uses a post-bisulfite adaptor tagging (PBAT) approach. Two methylomes need to be generated: a classic methylome following bisulfite conversion and analyzing both methylated and hydroxymethylated cytosines and a methylome analyzing only methylated cytosines, respectively. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocols have been successfully applied to different human samples and yield robust and reproducible results.

Kirschner et al. Multiplexing for oxidative bisulfite sequencing (oxBS-seq). Methods Mol Biol. 2018;1708:665-678. doi: 10.1007/978-1-4939-7481-8_34.
DNA modifications, especially methylation, are known to play a crucial part in many regulatory processes in the cell. Recently, 5-hydroxymethylcytosine (5hmC) was discovered, a DNA modification derived as an intermediate of 5-methylcytosine (5mC) oxidation. Efforts to gain insights into function of this DNA modification are underway and several methods were recently described to assess 5hmC levels using sequencing approaches. Here we integrate adaptation based multiplexing and high-efficiency library prep into the oxidative Bisulfite Sequencing (oxBS-seq) workflow reducing the starting amount and cost per sample to identify 5hmC levels genome-wide.

2017 oxBS papers:

Ma et al. Mouse olfactory bulb methylome and hydroxymethylome maps reveal noncanonical active turnover of DNA methylation. Epigenetics. 2017 Aug;12(8):708-714. doi: 10.1080/15592294.2017.1356958. Epub 2017 Sep 25.
Hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by TET enzymes presents a particular regulatory mechanism in the mammalian brain. However, although methylation and hydroxymethylation of cytosines in non-CpG contexts have been reported, these mechanisms remain poorly understood. Here, we applied TAB-seq and oxBS-seq selectively to detect 5hmC and 5mC at base resolution in olfactory bulb derived from female mice. We found that active turnover of 5mC to 5hmC occurred in both CpG and non-CpG contexts. Strikingly, we identified a different sequence preference for 5mC and 5hmC in a CH context, in which H = A, C, or T, TNCA/TC for 5mC and NNCA/T/CN for 5hmC. More importantly, we found that genes showing 5mC to 5hmC turnover showed only limited overlap in CpG and CH contexts, and that olfactory receptor genes were marked with higher turnover of 5mC to 5hmC in non-CpG context. Collectively, we identified an unexpected sequence preference for non-CpG hydroxymethylation and distinct target genes regulated by the turnover of 5mC to 5hmC in CpG and CH contexts.

Bhattacharyya et al. Altered hydroxymethylation is seen at regulatory regions in pancreatic cancer and regulates oncogenic pathways. Genome Res. 2017 Oct 6. doi: 10.1101/gr.222794.117
Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4 Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.

Spiers et al. 5-hydroxymethylcytosine is highly dynamic across human fetal brain development. BMC Genomics. 2017 Sep 18;18(1):738. doi: 10.1186/s12864-017-4091-x.
This study represents the first systematic analysis of dynamic changes in DNA hydroxymethylation (5hmC) across neurodevelopment and highlights the potential importance of this modification in the human brain. Epigenetic processes play a key role in orchestrating transcriptional regulation during the development of the human central nervous system. Of particular interest is 5hmC, a modification that is enriched in the human brain and hypothesized to play an important role in neuronal function, learning and memory. The authors quantified 5hmC across the genome of 71 human fetal brain samples spanning 23 to 184 days post-conception, identifying widespread changes in 5hmC occurring during human brain development, notable sex-differences in 5hmC in the fetal brain, and interactions between 5mC and 5hmC at specific sites. The resrearch also allowed the identification of loci where 5hmC in the fetal brain is associated with genetic variation. A searchable database of the fetal brain 5hmC data is available as a resource to the research community.

Mellén et al. 5-hydroxymethylcytosine accumulation in postmitoticneurons results in functional demethylation of expressed genes. Proc Natl Acad Sci U S A. 2017 Sep 12;114(37):E7812-E7821. doi: 10.1073/pnas.1708044114.
5-hydroxymethylcytosine (5hmC) occurs at maximal levels in postmitotic neurons, where its accumulation is cell-specific and correlated with gene expression. Here we demonstrate that the distribution of 5hmC in CG and non-CG dinucleotides is distinct and that it reflects the binding specificity and genome occupancy of methylcytosine binding protein 2 (MeCP2). In expressed gene bodies, accumulation of 5hmCG acts in opposition to 5mCG, resulting in “functional” demethylation and diminished MeCP2 binding, thus facilitating transcription. Non-CG hydroxymethylation occurs predominantly in CA dinucleotides (5hmCA) and it accumulates in regions flanking active enhancers. In these domains, oxidation of 5mCA to 5hmCA does not alter MeCP2 binding or expression of adjacent genes. We conclude that the role of 5-hydroxymethylcytosine in postmitotic neurons is to functionally demethylate expressed gene bodies while retaining the role of MeCP2 in chromatin organization

Garcia-Gomez et al. TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells. Nucleic Acids Research 20017, 29 July, gkx666, https://doi.org/10.1093/nar/gkx666
The plasticity of myeloid cells is illustrated by a diversity of functions including their role as effectors of innate immunity as macrophages (MACs) and bone remodelling as osteoclasts (OCs). TET2, a methylcytosine dioxygenase highly expressed in these cells and frequently mutated in myeloid leukemias, may be a key contributor to this plasticity. Through transcriptomic and epigenomic analyses, we investigated 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and gene expression changes in two divergent terminal myeloid differentiation processes, namely MAC and OC differentiation. MACs and OCs undergo highly similar 5hmC and 5mC changes, despite their wide differences in gene expression. Many TET2- and thymine-DNA glycosylase (TDG)-dependent 5mC and 5hmC changes directly activate the common terminal myeloid differentiation programme. However, the acquisition of differential features between MACs and OCs also depends on TET2/TDG. In fact, 5mC oxidation precedes differential histone modification changes between MACs and OCs. TET2 and TDG downregulation impairs the acquisition of such differential histone modification and expression patterns at MAC-/OC-specific genes. We prove that the histone H3K4 methyltransferase SETD1A is differentially recruited between MACs and OCs in a TET2-dependent manner. We demonstrate a novel role of these enzymes in the establishment of specific elements of identity and function in terminal myeloid differentiation.

Ancey et al. TET-catalyzed 5-hydroxymethylation precedes HNF4A promoter choice during differentiation of bipotent liver progenitors. Stem Cell Reports. 2017 Jul 11;9(1):264-278. doi: 10.1016/j.stemcr.2017.05.023.
Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

Marzi et al. A histone acetylome-wide association study of Alzheimer’s disease: neuropathology-associated regulatory variation in the human entorhinal cortex. bioRxiv preprint first posted online Sep. 1, 2017; doi: http://dx.doi.org/10.1101/183541.
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by the progressive accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles in the neocortex. Recent studies have implicated a role for regulatory genomic variation in AD progression, finding widespread evidence for altered DNA methylation associated with neuropathology. To date, however, no study has systematically examined other types of regulatory genomic modifications in AD. In this study, we quantified genome-wide patterns of lysine H3K27 acetylation (H3K27ac) – a robust mark of active enhancers and promoters that is strongly correlated with gene expression and transcription factor binding – in entorhinal cortex samples from AD cases and matched controls (n = 47) using chromatin immunoprecipitation followed by highly parallel sequencing (ChIP-seq). Across ~182,000 robustly detected H3K27ac peak regions, we found widespread acetylomic variation associated with AD neuropathology, identifying 4,162 differential peaks (FDR < 0.05) between AD cases and controls. These differentially acetylated peaks are enriched in disease-specific biological pathways and include regions annotated to multiple genes directly involved in the progression of Aβ and tau pathology (e.g. APP, PSEN1, PSEN2, MAPT), as well as genomic regions containing variants associated with sporadic late-onset AD. This is the first study of variable H3K27ac yet undertaken in AD and the largest study investigating this modification in the entorhinal cortex. In addition to identifying molecular pathways associated with AD neuropathology, we present a framework for genome-wide studies of histone modifications in complex disease, integrating our data with results obtained from genome-wide association studies as well as other epigenetic marks profiled on the same samples.

Lutz et al. Epigenetic Regulation of the Kappa Opioid Receptor by Child Abuse.Biol Psychiatry. 2017 Jul 27. pii: S0006-3223(17)31812-7. doi: 10.1016/j.biopsych.2017.07.012.
Experiences of abuse and neglect during childhood are major predictors of the emergence of depressive and suicidal behaviors throughout life. The underlying biological mechanisms, however, remain poorly understood. Here, we focused on the opioid system as a potential brain substrate mediating these effects. Postmortem samples from three brain structures regulating social bonds and emotions were analyzed. Groups were constituted of depressed individuals who died by suicide, with or without a history of severe child abuse, and of psychiatrically healthy control subjects. Expression of opioid peptides and receptors was measured using real-time polymerase chain reaction. DNA methylation, a major epigenetic mark, was investigated using targeted bisulfite sequencing and characterized at functional level using in vitro reporter assays. Finally, oxidative bisulfite sequencing was used to differentiate methylation and hydroxymethylation of DNA. A history of child abuse specifically associated in the anterior insula with a downregulation of the kappa opioid receptor (Kappa), as well as decreased DNA methylation in the second intron of the Kappa gene. In vitro assays further showed that this intron functions as a genomic enhancer where glucocorticoid receptor binding regulates Kappa expression, unraveling a new mechanism mediating the well-established interactions between endogenous opioids and stress. Finally, results showed that child abuse is associated in the Kappa intron with a selective reduction in levels of DNA hydroxymethylation, likely mediating the observed downregulation of the receptor. Altogether, our findings uncover new facets of Kappa physiology, whereby this receptor may be epigenetically regulated by stressful experiences, in particular as a function of early social life.

Hernandez Mora et al. Profiling of oxBS-450K 5-hydroxymethylcytosine in human placenta and brain reveals enrichment at imprinted loci. Epigenetics. 2017 Jul 5:0. doi: 10.1080/15592294.2017.1344803.
DNA methylation (5-methylcytosine, 5mC) is involved in many cellular processes and is an epigenetic mechanism primarily associated with transcriptional repression. The recent discovery that 5mC can be oxidized to 5-hydromethylcytosine (5hmC) by TET proteins has revealed the “sixth base” of DNA and provides additional complexity to what was originally thought to be a stable repressive mark. However, our knowledge of the genome-wide distribution of 5hmC in different tissues is currently limited. Here, we sought to define loci enriched for 5hmC in the placenta genome by combining oxidative bisulphite (oxBS) treatment with high-density Illumina Infinium HumanMethylation450 methylation arrays and to compare our results with those obtained in brain. Despite identifying over 17,000 high-confidence CpG sites with consistent 5hmC enrichment, the distribution of this modification in placenta is relatively sparse when compared to cerebellum and frontal cortex. Supported by validation using allelic T4 β-glucosyltransferase assays we identify 5hmC at numerous imprinted loci, often overlapping regions associated with parent-of-origin allelic 5mC in both placenta and brain samples. Furthermore, we observe tissue-specific monoallelic enrichment of 5hmC overlapping large clusters of imprinted snoRNAs-miRNAs processed from long noncoding RNAs (lncRNAs) within the DLK1-DIO3 cluster on chromosome 14 and SNRPN-UBE3A domain on chromosome 15. Enrichment is observed solely on the transcribed alleles suggesting 5hmC is positively associated with transcription at these loci. Our study provides an extensive description of the 5hmC/5mC landscape in placenta with our data available at www.humanimprints.net , which represents the most comprehensive resource for exploring the epigenetic profiles associated with human imprinted genes.

Benešová et al. DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas.Retrovirology. 2017 Mar 17;14(1):20. doi: 10.1186/s12977-017-0342-9.
Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors.We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

Kawasaki et al. Genome-wide mapping of 5-hydroxymethyluracil in the eukaryote parasite Leishmania. Genome Biol. 2017; 18: 23. doi: 10.1186/s13059-017-1150-1
5-Hydroxymethyluracil (5hmU) is a thymine base modification found in the genomes of a diverserange of organisms. To explore the functional importance of 5hmU, we develop a method for the genome-wide mapping of 5hmU-modified loci based on a chemical tagging strategy for the hydroxymethyl group. We apply the method to generate genome-wide maps of 5hmU in the parasitic protozoan Leishmania sp. In this genus, another thymine modification, 5-(β-glucopyranosyl) hydroxymethyluracil (base J), plays a key role during transcription. To elucidate the relationship between 5hmU and base J, we also map base J loci by introducing a chemical tagging strategy for the glucopyranoside residue. Observed 5hmU peaks are highly consistent among technical replicates, confirming the robustness of the method. 5hmU is enriched in strand switch regions, telomeric regions, and intergenic regions. Over 90% of 5hmU-enriched loci overlapped with base J-enriched loci, which occurs mostly within strand switch regions. We also identify loci comprising 5hmU but not base J, which are enriched with motifs consisting of a stretch of thymine bases. By chemically detecting 5hmU we present a method to provide a genome-wide map of this modification, which will help address the emerging interest in the role of 5hmU. This method will also be applicable to other organisms bearing 5hmU.

Do et al. Mechanisms and disease associations of haplotype-dependent allele-specific DNA methylation. Am J Hum Genet. 2016 May 5;98(5):934-955. doi: 10.1016/j.ajhg.2016.03.027.
Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A(∗)-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders.

Khamis et al. CpG traffic lights are markers of regulatory regions in humans. bioRxiv doi: https://doi.org/10.1101/095968
In this paper the authors demonstrate that single CpG methylation can serve as a more accurate predictor of gene expression compared to average promoter / gene body methylation. CpG positions with significant correlation between methylation and expression of a gene nearby (named CpG traffic lights) are evolutionary conserved and enriched for exact TSS positions and active enhancers. Among all promoter types, CpG traffic lights are especially enriched in poised promoters. Genes that harbor CpG traffic lights are associated with development and signal transduction. Methylation levels of individual CpG traffic lights vary between cell types dramatically with the increased frequency of intermediate methylation levels, indicating cell population heterogeneity in CpG methylation levels. Being in line with the concept of the inherited stochastic epigenetic variation, methylation of such CpG positions might contribute to transcriptional regulation. Alternatively, one can hypothesize that traffic lights are markers of absent gene expression resulting from inactivation of their regulatory elements. The CpG traffic lights provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to expression.

Gross et al. Gene-body 5-hydroxymethylation is associated with gene expression changes in the prefrontal cortex of depressed individuals. Transl Psychiatry. 2017 May 9;7(5):e1119. doi: 10.1038/tp.2017.93
The authors of this study report the first genome-wide analysis of 5hmC in the depressed brain. Consistent with previous global 5hmC analyses in other phenotypes, and likely owing to the inter-individual variability in 5hmC content, the distribution of 5hmC across chromosomes and genomic features was not different between prefrontal cortex of depressed and psychiatrically healthy controls. The authors did, however, find 550 CpGs with suggestive evidence of differential hydroxymethylation. Of these, the CpGs in the gene body of myosin XVI (MYO16) and insulin-degrading enzyme were validated using targeted oxidative bisulfite sequencing. Furthermore, the enrichment of 5hmC was also associated with changes in the expression of these two genes in depressed suicides. Together, the results present a novel mechanism linking increased 5hmC to depression and provide a framework for future research in this field.

Raiber et al. Base resolution maps reveal the importance of 5-hydroxymethylcytosine in a human glioblastoma. npj Genome Medicine. 2017; doi:10.1038/s41525-017-0007-6Importance of studying 5hmC and 5mC revealed in human glioblastomaUsing single base resolution maps of whole genomes, methylomes and hydroxylmethylomes for matched human glioblastoma and tumour margin samples delivers new insights into the interrelation of genetics and epigenetic variations. Previous studies using bisulfite converted DNA to measure combined levels of 5mC and 5hmC have led to genome-wide hypomethylation being regarded as an epigenetic hallmark of tumorigenesis. However, by using both bisulfite and oxidative bisulfite (oxBS) converted DNA to discriminate between 5mC and 5hmC, the authors identified global hypermethylation in the tumour. This result suggests that data obtained from bisulfite-only approaches need to be re-interpreted. Notably, the authors also observed that cells neighbouring tumour cells display epigenetic alterations characteristic of the tumour itself although genetically they appear “normal”. This shows the potential transfer of epigenetic information between cells that contributes to the intratumour heterogeneity of glioblastoma.

Zhang et al. Acetylation Enhances TET2 Function in Protecting against Abnormal DNA Methylation during Oxidative Stress. Molecular Cell. 2017; 65;2 -323 Acetylation of TET2 and binding to DMNT1 enhances protection against abnormal DNA methylationPrevious studies have revealed the function of the ten-eleven translocation (TET) family of proteins in DNA demethylation, now researchers from the Johns Hopkins University School of Medicine have further elucidated the mechanism by which the TET2 protein protects against abnormal DNA methylation in cancer cells. The researchers demonstrated that acetylation of TET2 increases enzymatic activity of the protein and enhances its interaction with DNA (cytosine-5)-methyltransferase 1 (DNMT1). The binding with DNMT1 further increases the stability of the TET2 protein and localises the protein to chromatin where it can prevent accumulation of DNA methylation by converting 5mC to 5hmC.

Chen et al. Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine. BMC Genomics. 2017; 18:96 A low-cost, adaptable method to quantify 5hmC and 5mC modificationsThis study is a proof-of-principle which describes a novel, low-cost, versatile methodology whereby bisulfite and oxidative bisulfite conversion of DNA can be used to quantify 5mC or 5hmC modifications at targeted genomic regions from a variety of samples. Samples were analysed from multiple types of disease and tissue, including post-mortem brain tissue and cancerous tumours. Using the TrueMethyl kit, the authors were able to analyse 5mC and 5hmC content in the samples in parallel. Bisulfite or oxidative bisulfite converted DNA was used in multiplex PCR reactions to amplify targeted genomic regions prior to next-generation sequencing. This approach could be widely applicable to, for example, validation of whole-genome studies, or to clinical or candidate gene studies.

Yotova et al. Epigenetic Alterations Affecting Transcription Factors and Signaling Pathways in Stromal Cells of Endometriosis. PLOS One. 2017 e0170859 Disease-associated changes in 5hmC modifications occur in genomic regions with transcriptional regulatory activityThis study investigated the epigenetic changes associated with endometriosis in human samples. Whole genome 5mC and 5hmC profiles were compared in healthy and diseased endometrial cells and stromal cells. 5hmC modifications were significantly reduced in the epithelial cells of diseased samples compared to normal samples; however 5hmC levels were comparable between diseased and normal stromal cells. Furthermore, no difference in 5mC was witnessed. This indicates that 5hmC changes, but not 5mC, are specific to the affected cell type in diseased samples. Targeted oxidative bisulfite sequencing, using the TrueMethyl kit, revealed that 5hmC modifications largely mirrored 5mC, and that these modifications reside within functional genetic loci and in regions controlling the expression of genes associated with endometriosis lesion formation.

2016 oxBS papers:

Li et al. Whole-genome analysis of the methylome and hydroxymethylome in normal and malignant lung and liver. Genome Res. 2016. 26: 1730-1741
The authors applied oxidative bisulfite sequencing to generate whole-genome DNA methylation and hydroxymethylation maps at single-base resolution in human normal liver and lung as well as paired tumor tissues. the research revealed 5hmC to be significantly enriched in CpG island (CGI) shores while depleted in CGIs themselves, especially in active genes. Hydroxymethylation on promoters, gene bodies, and transcription termination regions (TTRs) showed strong positive correlation with gene expression within and across tissues, suggesting that 5hmC is a marker of active genes and could play a role in gene expression mediated by DNA demethylation. Comparative analysis of methylomes and hydroxymethylomes revealed that 5hmC is significantly enriched in both tissue-specific DMRs (t-DMRs) and cancer-specific DMRs (c-DMRs), and 5hmC is negatively correlated with methylation changes, especially in non-CGI-associated DMRs. These findings revealed novel reciprocity between epigenetic markers at CGI shores corresponding to differential gene expression in normal tissues and matching tumors. Overall, the study provided a comprehensive analysis of the interplay between the methylome, hydroxymethylome, and histone modifications during tumorigenesis.

Gross et al. Variations in 5-methylcytosine and 5-hydroxymethylcytosine among human brain, blood, and saliva using oxBS and the Infinium MethylationEPIC array Biology Methods and Protocols 1 (1) 2016CEGX True Methyl kit is compatible with the Illumina Infinium MethylationEPIC array and 5hmC accounts for approximately one-third of methylation events in bisulfite sequencingDNA was extracted from human blood, brain and saliva and subject to bisulfite or oxidative bisulfite conversion, before analysis of 5mC and 5hmC modifications using the Illumina Infinium MethylationEPIC array. The TrueMethyl kit was used for oxidative bisulfite conversion and efficiency was directly comparable to that of the Zymo EZ DNA Methylation bisulfite conversion kit. 5mC and 5hmC modifications were distributed throughout the genome including regions with transcriptional regulatory activity. 5hmC accounts for approximately one-third of the total signal for bisulfite converted data; therefore it is crucial that this be considered for future work.

Solvsten et al. Voluntary Physical Exercise Induces Expression and Epigenetic Remodeling of VegfA in the Rat Hippocampus Molecular Neurobiology 1-16. 20165hmC modifications are detected in rat brain at promoter regions of key growth factorsIn this study, the aim was to characterise gene expression and accompanying epigenetic changes in rat hippocampus and frontal cortex in response to exercise over a period of two weeks. Expression of a panel of growth factor genes was analysed; several were upregulated in one or both of the brain areas. VegfA was upregulated in active rats in the hippocampus only, and this was mirrored by hypomethylation of CpG sites in the promoter region of the gene. Specifically, hypomethylation was observed in predicted Sp1/Sp3 transcription factor binding sites. Using oxidative bisulfite sequencing, 5hmC modifications were observed at a higher frequency than 5mC, but 5hmC levels were unaffected by exercise. This may suggest that 5hmC modifications are less dynamic than 5mC modifications in response to environmental factors such as exercise.

Johnson et al. 5-Hydroxymethylcytosine localizes to enhancer elements and is associated with survival in glioblastoma patients Nature Communications.7, Article number: 13177. 2016Functional implications of 5hmC on transcriptional regulation and glioblastoma patient survivalParallel bisulfite and oxidative bisulfite sequencing was performed in glioblastoma and prefrontal cortex tissue, and an algorithm utilised to estimate levels of 5mC, 5hmC or unmodified sites genome-wide. 5hmC modifications were significantly lower in glioblastoma tissue compared to normal tissue. 5hmC modifications were enriched in regions associated with transcription factor binding, in enhancer or super-enhancer elements, alternative mRNA splicing and in genes commonly mutated in glioblastoma. Furthermore, 5hmC modifications were enriched in actively transcribed regions indicating a positive correlation with expression. Patients who had tumours with lower 5hmC levels had a poorer prognosis, therefore 5hmC levels may have direct functional implications on survival.

Wallner et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics & Chromatin. 2016; 9:33.De-repression of a phagocytic gene network after onset of macrophage differentiationThe elucidation of monocyte to macrophage differentiation can be demonstrated by high-throughput sequencing and the investigation of DNA demethylation is an important part of this. DNA demethylation in monocytes affects only a small number of genes important in macrophage structure and function and is repressed in somatic tissue. TrueMethyl ox-BS sequencing kit was employed to differentiate 5mC from 5hmC and thus ascertain the level of 5hmC in monocytes. Levels of 5hmC are increased during the first 12 hours of differentiation. These increased levels are an indicator of TET enzyme activity which appears to be targeted to specific genomic regions unique to macrophages upon induction of differentiation by CSF1.

Vento-Tomo DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes Journal of Allergy and Clinical Immunology 139 (1) 2017Disease-associated demethylation of CpG sites is coupled to an increase in 5hmC and expression of inflammasome-related genes.Autoinflammatory syndromes can be caused by increased inflammasome activity due to gene mutations; however, the same mutations in different individuals do not necessarily result in similar phenotypes. During differentiation of macrophages from monocytes, or when monocytes were stimulated with pro-inflammatory cytokines, enzymatic demethylation of CpG sites was coupled to an increase in 5hmC at the same sites, as well as an increase in proximal gene expression. The CEGX TrueMethyl kit was used for oxidative bisulfite conversion of DNA. Demethylation was catalysed by TET2, and was found to be dependent on NFκB activity in this context. These changes in DNA methylation were also present in cryopyrin-associated periodic syndromes (CAPS) ex vivo patient samples, regardless of gene mutational status, and could be reversed by anti-IL-1 therapy.

Green et al. Hydroxymethylation is uniquely distributed within term placenta, and is associated with gene expression. FASEB J. 2016.5hmC modifications in placental tissue are associated with dynamically-expressed genes throughout placental maturation and development.The CEGX TrueMethyl kit was used to convert DNA in parallel from 23 placental tissue samples in order to quantify genomewide 5mC or 5hmC modifications using the Illumina HumanMethylation450 BeadChip. 19 of these samples were also used for RNA-seq analysis, before combining the data to measure the relationship between 5mC, 5hmC and gene expression. Although mean 5hmC levels were lower than 5mC, a high number of probes showed robust levels of 5hmC across samples. CpG islands were distinctly devoid of 5hmC, regions with H3K27Ac were depleted of 5hmC, but poised enhancer regions were enriched in 5hmC. 5hmC modifications correlated significantly with the expression of transitional genes important for placental maturation and development.

Hadad et al. Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice. Epigenetics & Chromatin. 2016; 9:30Contradicting the genomic hypomethylation theory of agingThis study aimed to investigate aging associated DNA methylation. Total levels of 5mC or 5hmC in mouse brains did not change with aging in either male or females; however, oxBS-Seq using the TrueMethyl kit allowed more localised methylation changes to specific genomic loci to be detected. Differential methylation levels were also reported between the sexes. This study demonstrates the need for careful study design ensuring base-specific analysis of specific tissue and sexes.

Toraño et al. Age-associated hydroxymethylation in human bone-marrow mesechymal stem cells. J. Transl Med. 2016; 14:207Epigenetic modifications in bone-marrow suggest that the age of donors should be consideredThis study aimed to investigate age-associated DNA hydroxymethylation. Levels of 5hmC in mesechymal stem cells, determined using the MethylBlue ox-BS protocol, indicated that CpG sites gained hydroxymethylation and lost 5mC in the advanced age group. This suggests a role for 5hmC during aging. This study also noted that there were age-associated patterns of 5hmC changes in different age groups but none were locus-specific. This research demonstrates that 5hmC should be considered with 5mC as an important mark in the study of DNA methylation and aging. These marks may also affect the potential of the bone-marrow for therapeutic use.

Lunnon et al. Variation in 5-hydroxymethylcytosine across human cortex and cerebellum. Genome Biol. 2016; 17:27The incorporation of oxidative bisulfite treatment helps quantify 5hmC in the human cortex and cerebellumThis study was designed to quantify and compare DNA methylation in human cortex and cerebellum tissue. The treatment of samples using TrueMethyl oxBS in conjunction with standard BS treatment allowed discrimination between 5mC and 5hmC. This enabled the accurate measurement of 5hmC at functional sites within the tissue, the demonstration of differences between the prefrontal cortex and the cerebellum and to show that there is considerable inter-individual variation of 5hmC at some sites. It is possible that these new data could confound previous findings attributed to DNA variation in methylation using cumulative 5mC and 5hmC values. It is anticipated that these data could be used to investigate the role of 5hmC in neurological disorders.

Heyn et al. Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer. Genome Biol. 2016; 17:11Super-enhancers targeted by aberrant DNA methylationThis study investigated the epigenic alterations caused by aberrant DNA methylation of super-enhancers associated with cancer. The examination of DNA methylation as a chemical mark in gene regulation showed disease-associated variation at super-enhancer sites when compared to normal tissue. Comparison of oxBS and BS treated samples showed that 5hmC was not responsible for the increase in DNA methylation observed at these sites in any of the cancer samples. Super-enhancer DNA methylation profiles appear to be altered, possibly by changes in transcription factor binding, with subsequent effects on gene expression. There is a need for more extensive catalogues of human DNA methylomes to improve the understanding of DNA methylation at multiple sites.

Yue et al. Control of Foxp3 stability through modulation of TET activity. J Exp Med 2016; Vol 213 (3) 377-397Regulatory T cell development is controlled by methylation status at key regulatory regions and can be enhanced by TET activating agents.Foxp3 expression is regulated by methylation status at intragenic regulatory elements and is an essential regulator of regulatory T cell (Treg) development and function: key to preventing autoimmunity and maintaining immune homeostasis. Two TET isoforms (Tet2/Tet3) are responsible for demethylation of the Foxp3 regulatory locus which results in stabilisation of Foxp3 expression and maintenance of Treg cell identity. Bisulfite and oxidative bisulfite sequencing, using the CEGX TrueMethyl kit, revealed loss of 5mC and an increase in 5hmC at the regulatory locus during Treg development which was impaired in Tet2/Tet3 double knockout mice. 5hmC was enriched in Treg precursor cells. Vitamin C, an agonist of the TET proteins, was able to enhance demethylation and stabilise Foxp3 expression during Treg cell development thus potentially providing a novel therapeutic strategy to combat immune system dysfunctions.

Vento-Tormo et al. IL-4 orchestrates STAT6-mediated DNA demethylation leading to dentritic cell differentiation.Genome Bioloy 2016; 17:4Functional implications of 5mC loss, via the 5hmC intermediate, on the monocyte differentiation process in response to stimulation.Differentiation of innate immune system component cells from progenitors is a tightly regulated process, with epigenetic modifications playing a central role in translating extracellular signals into control of cell identity and fate. In an in vitro model of monocyte differentiation to macrophages or dendritic cells, the differentiation process induced demethylation of several thousand CpG sites which were cell-type specific and enriched in enhancer regions. Changes in methylation status had functional consequences: several thousand genes were either up- or down-regulated. TET-mediated demethylation events were confirmed by the formation of the 5hmC intermediate as determined using the CEGX TrueMethyl kit. Changes in the epigenome were reliant upon an intact JAK2-STAT6 signalling pathway, thus demethylation is a crucial component of the cellular response to differentiation signals.

2015 oxBS papers:

Wille et al. 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch. PNAS 20155hmC modification of epithelial cell and Epstein-Barr virus genomes can regulate lytic viral activation and tumour formation
Epstein-Barr virus has been associated with the formation of undifferentiated nasopharyngeal carcinoma (NPC) from undifferentiated epithelial cells, and the EBV viral genome can undergo methylation changes which determine the type of infection (either lytic or latent). Using oxidative bisulfite sequencing, 5hmC modification of the viral genome was detected and shown to accompany lytic reactivation of the virus. Furthermore, 5hmC modification accumulated during normal epithelial cell differentiation but not in EBV infected NPC cells. Increasing or decreasing TET activity resulted in increased 5hmC modification, or increased 5mC modification, of lytic EBV promoter regions in EBV infected cell lines, respectively. 5hmC modification of the promoters altered binding of key transcription factors which regulate the EBV switch from latent to lytic infection. Altered levels of 5hmC in the host and viral genome may contribute to the formation of NPC tumour cells.

Page et al. Hepatic Stellate Cell Transdifferentiation Involves Genome-Wide Remodeling of the DNA Methylation Landscape. Journal of Hepatology 2015Disease-specific modifications in 5mC/5hmC drive hepatocyte differentiation and fibrosis
Liver fibrosis results from a phenotypic change from hepatic stellate cells to a pro-fibrogenic myofibroblast-like cell, a transdifferentiation accompanied by a huge change in the transcriptome and thought to be regulated by epigenetic changes. Using the CEGX TrueMethyl oxidative bisulfite kit, liver fibrosis was shown to be accompanied by a stabilisation of 5mC, depletion of global 5hmC levels and altered expression of the TET and DNMT enzymes. 5hmC modifications were altered across the genome between normal and disease-like cells, however a particular enrichment in 5hmC modification was found on chromosome 9. Depletion of Dnmt3a resulted in decreased expression of collagen 1A1 and α-smooth muscle actin; thus changes in the epigenetic landscape, including disease-specific 5mC/5hmC modifications, control differentiation of hepatocytes and drive fibrogenesis.

Moran et al. Validation of a DNA methylation microarray for 850,000 CpG sites of the human genome enriched in enhancer sequences. Epigenomics 2015The MethylationEPIC BeadChipInfinium 850K array is compatible with the CEGX TrueMethyl kit for genome wide detection of 5hmC
5mC is the most well-characterised epigenetic modification and its importance in the regulation of gene expression is highlighted by the distorted DNA methylation patterns witnessed in numerous diseases. The Illumina Infinium HumanMethylation450 BeadChip was previously the most widely used platform to interrogate the DNA methylome; 5mC and 5hmC patterns could be mapped at 450,000 CpG sites across the genome. In this study, the 450K and the new MethylationEPIC BeadChip Infinium 850K arrays from Illumina were compared, with special focus on the 333,265 CpG sites located in predicted or experimentally determined enhancer regions. The 850K array can reproduce the 450K data reliably, with Pearson correlation coefficients of > 0.99, and can be used for assessment of modifications from fixed and embedded samples. Ability to detect 5hmC levels across the genome was confirmed using the CEGX TrueMethyl kit.

Mendioroz et al. Trans effect of chromosome aneuploidies on DNA methylation patterns in human Down syndrome and mouse models. Genome Biology 2015, 16:263Altered transcription factor binding due to epigenetic changes in brain cells and T cells; associated with trisomy 21 and with functional implications on gene expression
Epigenetic alterations have recently been proposed as a mechanism controlling the spectrum of phenotypes associated with chromosomal aneuploidy and Down syndrome, where the additional chromosome may impact upon cell networks in trans, and lead to epigenetic alterations. DNA 5mC and 5hmC content was quantified using the Illumina 450K Methylation BeadChip and validated using bisulfite sequencing, using the CEGX TrueMethyl kit for oxidative bisulfite conversion of DNA. Gene- and tissue-specific alterations in DNA methylation were identified in Down syndrome brain cells and T cells, where some differentially methylated regions harboured 5mC and 5hmC changes, and some had primarily 5hmC changes (enrichment in genes associated with brain development and function or T cell development). Altered DNA methylation patterns affecting developmentally-regulated genes were present in foetal samples, indicating early onset of changes, and a subset of these genes displayed altered expression in Down syndrome brain samples which was likely due to altered transcription factor binding.

Sellars et al. Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages. Nature immunology (July 2015)T cell fate is determined by the methylation or demethylation, via 5hmC, of a CD4 enhancer element which regulates gene expression and differentiation
The process of T cell development is highly regulated and involves heritable epigenetic programing of lineage specific genes in order to silence or enhance their expression accordingly. A shRNA screen revealed that Dnmt1 played a critical role in the regulation of CD4 expression and maintained effective silencing of the gene in cytotoxic T cells, and DNA methylation was confirmed as crucial to this process as Dnmt-depleted cells transferred to mice lost their CD4-silencing ability and expressed both CD4 and CD8 markers. A differentially methylated locus was established proximal to the CD4 transcription start site and was hypermethylated in CD8 cytotoxic T cells relative to CD4 helper T cells, and was present in T cell progenitors thus methylation at this locus determines cell fate. The TET enzymes mediate demethylation of this region at specific CpG sites in order to enhance CD4 expression, which was confirmed by the presence of 5hmC modifications. T cell differentiation is therefore dependent on epigenetic modification of the CD4 locus to modulate lineage-specific gene expression.

Kubo N et al. DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis. BMC Genomics. (2015)16:624Dynamic DNA methylation and hydroxymethylation modifications describe the phases of male germ cell development
The epigenetic landscapes and gene expression profiles of the various stages of spermatogenesis have been largely understudied, thus whole-genome bisulfite sequencing and RNA-sequencing was carried out in prospermatogonia (PSG) and spermatogonia (SG). The CEGX TrueMethyl kit was used to analyse 5mC and 5hmC. Large differences in global 5mC levels were witnessed between the developmental stages, with large (<12 Mb) regions of hypomethylation detected in both stages. Quantification of 5hmC confirmed that satellite repeats at the centromeric and periocentromeric regions were enriched for this modification in PSG, however 5hmC was only found at 1.4% of CpGs across the PSG genome. 5hmC modification is absent after this developmental stage, which may suggest that 5hmC modification is involved in the transcriptional silencing of these regions at this specific stage of sperm development.

Matsubara et al. Exploration of hydroxymethylation in Kagami-Ogata syndrome caused by hypermethylation of imprinting control regions. Clinical Epigenetics. (2015)7:905hmC is present at low levels in blood DNA extracted from Kagami-Ogata patients but may play a role in neurodevelopmental abnormalities
Kagami-Ogata syndrome is a human imprinting disease which has previously been described as a result of hypermethylation of two differentially methylated regions (DMRs) which act as imprinting control regions (ICRs): IG-DMR and MEG3-DMR. Using the CEGX TrueMethyl kit to bisulfite or oxidative bisulfite convert DNA, hypermethylation at the two DMRs was seen in DNA isolated from the blood of Kagami-Ogata syndrome patients but not in controls. This hypermethylation consisted almost exclusively of 5mC modifications rather than 5hmC. Higher levels of 5hmC were witnessed in neural tissue at both the genomic and DMR levels. Thus, 5hmC may play a role in the neurodevelopment problems of Kagami-Otaga patients and is consistent with 5hmC modifications being highly disease and tissue specific.

Field SF et al. Accurate Measurement of 5-Methylcytosine and 5-Hydroxymethylcytosine in Human Cerebellum DNA by Oxidative Bisulfite on an Array (OxBS-Array). PLoS One. 2015 Feb 23;10(2):e0118202Improving the sensitivity of 5hmC detection using multiple replicates and oxidative bisulfite conversion of DNA with the CEGX TrueMethyl kit
The established method of detecting DNA methylation (5mC) is to bisulfite convert samples prior to downstream analyses; however, this method cannot distinguish between 5hmC and 5mC and so bisulfite converted samples actually measure the combined modification of 5mC+5hmC. This study describes a method by which 5hmC modifications can be reproducibly detected using four replicates of bisulfite converted and four replicates of oxidative bisulfite converted cerebellum DNA. Four replicates allowed detection of 5hmC at over 114,000 probes on the Illumina 450K methylation array, however reduction of replicates to 2 allowed for reliable detection of 5hmC at only approx. 50,000 probes. Findings were validated using multiple approaches: LC-MS and qPCR with glucosylation of 5hmC sites and digestion with a restriction enzyme. This study highlights the importance of using multiple technical replicates to improve reproducibility and that 5hmC can be detected at extremely low levels.

2014 oxBS papers:

Stewart S et al. oxBS:-450K: A method for analysing hydroxymethylation using 450K Bead Chips. Methods. 2014 Aug 28Oxidative bisulfite conversion using the CEGX TrueMethyl kit can be combined with the Illumina Infinium HumanMethylation 450K BeadChip to quantify 5hmC levels
The Illumina Infinium HumanMethylation 450K BeadChip has been a well-used platform for detecting and quantifying DNA methylation levels, however the method of bisulfite conversion used in DNA preparation cannot distinguish between 5mC and 5hmC modifications. Using the CEGX TrueMethyl kit for oxidative bisulfite conversion of human brain DNA, 5hmC modified CpG sites can be differentiated from 5mC modified bases. 5hmC was detected in relatively high abundance in three brain DNA samples and at lower levels in blood DNA. 5hmC levels were enriched within gene bodies but depleted from promoter regions and CpG islands. 5hmC levels determined by the 450K BeadChip were validated using LC-MS and oxidative bisulfite pyrosequencing: 5hmC levels were found to vary by up to 10% between the array and pyrosequencing methods. Oxidative bisulfite conversion coupled with the 450K BeadChip array is therefore a reliable and accurate method to determine 5hmC levels across the genome.

Booth MJ et al. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nature Chemistry. 2014 Mar 23An in-depth step-by-step guide to oxidative bisulfite sequencing for the quantitative detection of 5hmC at single-base resolution
This study provides an in-depth, technical and comprehensive guide to carrying out oxidative bisulfite sequencing, which is an improved method for detecting 5hmC levels in a quantitative manner at single-base resolution and distinguishes this modification from 5mC. Development, potential applications, comparisons with other techniques and limitations of oxidative bisulfite sequencing are discussed in this paper. Information relating to experimental design, reagents and materials and troubleshooting are included. A step-by-step protocol with critical tips and instructions are given along with estimates of time consumption and anticipated results. This protocol recommends the use of the Cambridge Epigenetix TrueMethyl kit during the oxidative bisulfite conversion of DNA due to the optimisation of conditions, consistency and reproducibility that non-experts would benefit from.

Nakashima H et al. Effects of dppa3 on DNA methylation dynamics during primordial germ cell development in mice. Biol Reprod. 2013 May 23;88(5):125.Imprinted genes and retrotransposons are protected from TET-mediated demethylation during germ cell developmentWhole-genome demethylation is thought to occur at various stages during pre- and post-implantation development of the embryo, and is a highly dynamic process regulated by the TET enzymes and diluted during DNA replication. Dppa3 is thought to bind to and protect 5mC from conversion to 5hmC in the maternal genome; however the role of Dppa3 during further development is unknown. Using Dppa3-deficient mice and the CEGX TrueMethyl kit for oxidative bisulfite or bisulfite sequencing, DNA methylation patterns were shown to be altered at specific regions known to be important for silencing of imprinted genes and retrotransposon elements. Increased levels of 5mC were coupled to lower levels of 5hmC, thus Dppa3 likely plays a role in TET-mediated demethylation of these regions. These changes had no detectable impact on cell differentiation, however.

2012 oxBS papers:

Booth MJ et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science. 2012 May 18;336(6083):934-7.First description of how parallel sequencing can be used to quantify 5mC and 5hmC at single-base resolution
Many methods have been used to attempt to measure 5hmC modifications however no method described thus far can give quantitative and high-resolution information about this epigenetic mark. This study describes for the first time the application of bisulfite and oxidative bisulfite sequencing of samples in parallel before next generation sequencing in order to identify CpG sites which carry 5-methylcytosine or 5-hydroxymethylcytosine modifications. Oxidative bisulfite sequencing can specifically measure 5hmC, while bisulfite sequencing measures both 5mC and 5hmC combined. This novel approach was used to describe the epigenetic landscape in mouse embryonic stem cells and its sensitivity was verified using mass spectrometry. 5hmC modifications were enriched in the promoters of genes associated with transcription factor activity such as the Homeobox genes, and in intergenic regions with moderate levels of 5mC which may suggest that these regions are particularly susceptible to epigenetic modification and highlights the functional importance of 5hmC.